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Peroxisome proliferator-activated receptors (PPARs)

activation leading to reproductive toxicity in rodents

Małgorzata Nepelska

DISCLAIMER: This presentation and its contents do not constitute an official position of the European Commission or any of its services. Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use which might be made of this presentation or its contents

Adverse Outcome Pathways: From Research to Regulation

September 3-5, 2014

At the beginning

We had an AIM To develop a strategy for building a MoA based chemical category

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How?

toxicity

f(MoA)=

f( ) = chemical structure

Building MoA-based chemical category for toxicity prediction STEP 1. Chose endocrine active, data rich chemicals STEP 2. MoA matrix display of experimental data STEP 3. Mechanistic "blueprint" of phthalates STEP 4. Search for mechanistic analogues (other chemicals that

have similar MoA)

Phthalates ER PPAR AR AhRSertoli cells

spermatogenesis

Leydig cells

Decreased testosterone

stereodogenesis

oestrus cycle

Male reproductive

tract

Sperm parameters

Decreased AGD

DEP 0 1 1 1 0 0 / 0DiBP 1 1 1 1 1 1 1 1 1DPP 0 1 1 1 1 1 1 1

DCHP 0 0 1 1 1 1 1 1 1 1 1DHP 0 / 1 1 1 1 1 1 1 1DINP 0 0 0 1 1 1 1 1 0 1 /DIDP 0 0 1 0 0 / 1 0

DnOP 0 1 0 0 /

BBP 1 1 1 1 1 1 1 1 1 1 1

DprP 1 1 1

MEHP 1 1 1 1 1 1 1 1 1 1

DEHP / 1 / 1 1 1 1 1 1 1 1 1

KE MIE AO

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PPAR activation leading to reproductive toxicity in rodents

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PPAR activation

Hormone synthesis cholesterol

transport Hormone

levels Reproductive toxicity

MIE KE Adverse Outcome AOP-linked chemical initiators

PPARs peroxisome proliferator-activated receptors

family comprises the types α, γ and β/δ are nuclear receptor superfamily of

transcription factors that respond to specific ligands

regulate lipid and carbohydrate metabolism embryonic and foetal development cholesterol uptake and transport represent a potential molecular link between

reproductive function and carbohydrate and lipid metabolism

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MIE

PPARα,γ β/δ regulated genes

Xenobiotics Endocrine disruptors

Nutrients Fatty acids

Growth factors

inflammatory responses

embryonic and foetal development

intracellular trafficking of lipids

vascular functions

Lipids and carbohydrates metabolism

PPAR activation: evidence

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MIE MIE

Chemical initiator

In vitro binding

in vitro transactivation

Knock-out/inhibition/increased expression

DEHP - + Experiments with PPARα-null mice indicate involvement of the receptor in reproductive toxicity of phthalates

MEHP + + Inhibition studies

BBP +/- +

To be verified

DBP +/- +

To be verified

Bisphenol A -

+

Increased expression PPARγ

Butylparaben - + Increased expression PPARγ

Altered steroidogenic pathway

aromatase

testosterone

estradiol

estrone

Chemical initiator

PPAR binding&activation

17β-HSD IV

StAR

TSPO cholesterol

pregnenolone

P450scc

3 β -HSD-III

cholesterol

progesterone

androstenedione

aromatase

KE

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KE KE AO

Chemical Initiator

Decreased testosterone levels

Malformation of reproductive organs Testicular toxicity

DEHP

+ (Howdeshell et al.,

2008)

+ (Gray et al., 2000)

(Parks, 2000)

+ (Kwack et al., 2009)

BBP

+ (Howdeshell et al.,

2008)

+ (Gray et al., 2000)

(Nagao et al., 2000)

+ (Gray et al., 2000)

DBP

+ (Howdeshell et al.,

2008) (Barlow et al., 2003) (Mylchreest, 2000)

+ (Barlow et al., 2003) (Mylchreest, 2000)

+ (Mylchreest, 2000)

Bisphenol A

+ (Tanaka et al., 2006)

(Nakamura et al., 2010)

(Talsness et al., 2000)

+/- (Takagi et al., 2004)

(Kobayashi et al., 2002) (Talsness et al., 2000) (Tinwell et al. 2002)

+ (Talsness et al., 2000)

Butyl paraben

+ (Zhang et al., 2014)

+ (Zhang et al., 2014)

+ (Oishi et al., 2001)

+ effect present / no change ? no information *testosterone production

PPAR activation leading to reproductive toxicity in rodents

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PPAR activation

Hormone synthesis

cholesterol transport to

mitochondria Hormone

levels Reproductive toxicity

MIE KE Adverse Outcome

Malformation of reproductive organs Decreased AGD Hypospadias

Altered oestrus cycle Decreased ovary weight

PPAR activation leading to reproductive toxicity in rodents

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PPAR activation

Estradiol synthesis

cholesterol transport to

mitochondria

Hormone levels Reproductive

toxicity

PPAR activation

testosteronesynthesis

cholesterol transport to

mitochondria Hormone

levels Reproductive malformations

PPAR activation

Estradiol synthesis

cholesterol transport to

mitochondria Hormone

levels Altered estrus cycle

PPAR activation

testosteronesynthesis

cholesterol transport to

mitochondria Hormone

levels Testicular toxicity

AOP 1

AOP 2

AOP 3

pre

nta

l ad

ult

PPAR activation leading to reproductive toxicity in rodents

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testosteronesynthesis Hormone

levels Testicular toxicity

pre

nta

l ad

ult

PPARα/γ activation

testosteronesynthesis Hormone

levels Malformations Reproductive

organs

PPAR activation

Estradiol synthesis

cholesterol transport to

mitochondria Hormone

levels Altered estrus cycle

PPARα activation leading to reproductive tract malformations in males upon in utero exposure

Testosterone levels

PPARα activation

Testosterone synthesis

cholesterol transport to

mitochondria

MalformationReproductive

organs

MIE KE Adverse Outcome

Decreased AGD Hypospadias

AOP-linked chemical initiators

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PPARα activation leading to reproductive tract malformations in males upon in utero exposure

Key Events Experimental Support Strength of Evidence

Molecular Initiating Event: Binding to and activation to PPARα

DEHP/MEHP, BBP, DBP binding to PPARα in vitro, in silico PPARα transactivation by DEHP/MEHP, BBP, DBP,

butylparaben Experiments with PPARα-null mice indicate involvement

of the receptor in reproductive toxicity of phthalates

Moderate

Key Event: Impaired steroidogenesis

Impaired transport of cholesterol to mitochondria decreased gene expression of SR-B1, TSPO (PBR), StAR decreased gene expression of P450scc, 3β-HSD, 17β-HSD

Moderate

Key Event: Decreased testosterone levels

Decreased testosterone levels measured in plasma Decreased testosterone production measured ex-vivo

Strong

Adverse Outcomes: Reproductive tract malformations

DEHP, DBP,BBP, butylparaben, decreased AGD DEHP, DBP,BBP, Hypospadias

Strong

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Weak

Moderate

Strong

Challenges for these AOPs

Data mining

Literature organisation and structural capturing of the biological events

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Challenges for these AOPs cd.

Data mining

Literature organisation and structural capturing of the biological events

Quality and quantity of data in literature (PPAR α or/and γ), dose levels, more mechanisms involved

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Relevance for humans Mode of action PPAR expression Steroidogenesis is conserved

Adversity TDS- Testicular Dysgenesis Syndrome in humans

Future plans

To insert quantitative data into the OECD AOP-Knowledge Base

To further substantiate AOP with evidence from other chemicals

To develop other pathways interconnected with the current ones

aiming at AOP network

To further develop the database for capturing the literature and

provide a template for structured data gathering

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Acknowledgment

Brigitte Landesmann Edward Carney Sharon Munn Andrew Worth Julien Burton Alfonso Lostia

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Thank you

for coming questions